New biocatalysts from environmental gene libraries
01 / 2006 - unknown
The metagenome as a source of useful enzymes The environmental metagenome (i.e. the combined genomes of all microorgansms in a certain environment) constitutes a vast and almost unexplored pool of new genes and, consequently, potential new biocatalysts. A rapid way to harvest new biocatalysts is via the construction of gene banks that are constructed and expressed in E. coli and the screening of the E. coli recombinant libraries for new biotransformation genes. We have constructed various of such environmental gene banks in E. coli using DNA from microorganisms indigenous to different types of soil and sediment. The expression libraries were subsequently screened for clones encoding amidase and penicillin acylase activity. In one case, DNA was isolated from soil without an intermediate cultivation step to preserve a high degree of genetic diversity, which was demonstrated by DGGE analysis to be present in the starting material. In other cases, to reduce the number of clones required to find the actual target enzymes, DNA was also isolated from enrichment cultures, in which different aromatic and non-aromatic amides were supplied as sole nitrogen source. Amplified gene libraries were searched, using a three-step screening procedure that started with growth selection of amidase-expressing clones, followed by colorimetric screening of positives in microtiter plates, and a final round of HPLC measurement of the performance of selected clones in â-lactam antibiotic synthesis. This procedure resulted in the isolation of 7 new amidases including one with penicillin acylase activity. A new penicillin acylase Sequence analysis of the novel penicillin acylase (PAS2) revealed 83% identity with a recently cloned penicillin acylase of Flavobacterium sp. 650 and about 40-50% amino acid identity with the corresponding proteins of E. coli, Proteus rettgeri, Kluyvera cryocrescens, and Alcaligenes faecalis. Compared to the extensively studied penicillin acylase of E. coli ATCC 11105, the PAS2 enzyme showed superior potential for the synthesis of 6-aminopenicillanic acid derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed with the new enzyme than with E. coli penicillin acylase, making PAS2 an interesting candidate for biocatalytic application. The activity of this enzyme has been further improved by structure-inspired semirandom mutagenesis (Gabor and Janssen, 2004). Library size and chance of success The exploitation of the metagenome for novel biocatalysts by functional screening is dependent on the ability to express the target genes in a surrogate host. The probability to recover a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. We have derived a set of formulas that describe the chance to isolate a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion, and expression as a translational fusion. Genes of the latter category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analyzed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. The results revealed significant differences between taxonomic groups, and suggest that about 40% of the enzymatic activities may be recovered by expression cloning in E. coli (Gabor et al., 2004b).